Spinal with Diffuse Arachnoidal Dissemination in a Japanese Black Heifer Kazuyuki UCHIDA, Miki MURANAKA, Takayuki MURAKAMI1), Ryoji YAMAGUCHI and Susumu TATEYAMA Departments of Veterinary Pathology and 1)Veterinary Anatomy, Faculty of Agriculture, Miyazaki University, Miyazaki 889Ð2192, Japan (Received 9 June 1999/Accepted 16 August 1999)

ABSTRACT. A gelatinous focus with cystic spaces, was found in the posterior funiculus of the 2nd to 3rd lumbar levels of the of a Japanese Black heifer, 2 years old, with clinical signs of severe dysstasia. Histopathological examination revealed that the spinal lesion consisted of multifocal and diffuse proliferation of round cells with abundant vacuolar cytoplasm and hyperchromatic nuclei. In the lesions there was a number of cystic spaces containing aggregates of small round cells. The neoplastic foci showed a honeycomb structure divided by thin blood vessels, representing typical lesions of oligodendroglioma. Diffuse and multifocal proliferation of these round cells were also recognized in the subarachnoidal space in the sacral spinal cord. Immunohistochemically, the proliferating round cells were negative for glial fibrillary acidic protein. Based on these morphological features, the case was diagnosed as lumbar spinal oligodendroglioma with diffuse arachnoidal dissemination.—KEY WORDS: bovine, oligodendroglioma, spinal cord. J. Vet. Med. Sci. 61(12): 1323Ð1326, 1999

Oligodendroglioma is a common of animals, cord were also stained with Luxol fast blue (LFB), Masson’s especially dogs [13], but only one bovine case has been trichrome, Watanabe’s silver impregnation, and alcian blue reported [1]. Although both animal and human (pH 2.5). Histopathological examination revealed that the are known to occur predominantly in lumbar spinal lesion consisted of multifocal and diffuse the white matter of the cerebral hemispheres [9, 12, 13], proliferation of neoplastic cells with cystic spaces (Fig. 2). spinal oligodendrogliomas are also recognized occasionally The tumor mass was located mainly in the white matter, in children, and their clinical and pathological natures have and the posterior funiculus were almost totally replaced by been well demonstrated in several human cases [2]. In the proliferating tumor cells and cystic places. The contrast, spinal cord tumors including oligodendrogliomas neoplastic foci showed a honeycomb structure with solid [5, 15] are rare in animals, except for extra-medullar tumors proliferation of round cells, representing the typical such as spinal nephroblastomas in young dogs [13], and appearance of oligodendroglioma (Fig. 3). Most neoplastic bovine spinal are considered to be extremely cells were round in shape with abundant clear cytoplasm unusual [1]. and hyperchromatic central nuclei. The presence of The present paper describes the morphological and cytoplasmic processes was not confirmed. In the tumor immunohistochemical features of a spinal there were a number of blood vessels with thin vascular oligodendroglioma in a 2-year-old Japanese Black heifer walls, while the endothelial cells did not show apparent and discusses the similarity between human and bovine hyperplastic or proliferative changes. There were also a cases. few of well differentiated with well defined A 2-year-old Japanese Black heifer suddenly developed eosinophilic cytoplasmic processes in the neoplastic foci. hind limbs and dysstasia. The animal was treated In contrast, at the periphery of the neoplastic foci, diffuse by clysis with Ringer’s solution, but the clinical signs did proliferation of these astrocytes was prominent. Within the not improve. The animal was euthanatized by electric shock cystic spaces, there were aggregates of small number of 7 days after clinical onset at the owner’s request, and small round cells mimicking lymphocytes, with small necropsied immediately. In the brain, there were no hyperchromatic round nuclei and scanty cytoplasm. Mitoses significant lesions, but congestion and yellowish of the neoplastic cells were extremely rare. The neoplastic discoloration was recognized in the posterior area of the cells invaded the leptomeninges, and the subarachnoidal 2nd to 3rd levels of the lumbar spinal cord. The cut surface space was filled by a proliferation of these cells. Diffuse in this area revealed a pale gelatinous focus, approximately and multifocal proliferation of the round cells and 1 cm in diameter, with multiple cystic spaces filled with lymphocyte-like small round cells, were also seen in the clear fluid, located mainly in the posterior funiculus (Fig. subarachnoidal space in the 2nd and 3rd levels of the sacral 1). The gelatinous focus was attached to the , spinal cord (Fig. 4). In this area, some foci consisted of and the subarachnoidal space was replaced by the gelatinous aggregates of neoplastic cells, and some areas showed an material. In the subarachnoidal space of the lower lumbo- organoid structure made up of tumor cells, a few astrocytes sacral spinal cord, a small amount of fibrinous material, and their cytoplasmic processes (Fig. 5). Besides the spinal pale to tan in color, was distributed diffusely and widely. cord there was no evidences for of the spinal Tissue samples were fixed in 10% formalin and routinely cord tumor. processed. Paraffin sections were stained with hematoxylin Immunohistochemistry was performed using the Envision and eosin (HE), and some selected sections of the spinal polymer method (Dako Japan Co. Ltd., Kyoto, Japan). The 1324 K. UCHIDA ET AL.

Fig. 1. A gelatinous foci with cystic spaces mainly in the posterior funiculus of the lumbar spinal cords (L2 to L3). Fig. 2. Diffuse proliferation of neoplastic cells with forming many cystic spaces in the posterior funiculus of the lumbar spinal cord (L2). HE. × 10. Fig. 3. A honeycomb structure with accumulation of round neoplastic cells with a proliferation of blood vessels. HE. × 200. Fig. 4. Multiple foci (arrows) consisting of a proliferation of neoplastic cells in the subarachnoidal space of the sacral spinal cord (S3). HE. × 50. Fig. 5. High power magnification of Fig. 4. Neoplastic focus showing organoid structures. HE. × 100. Fig. 6. A few number of GFAP-positive astrocytes (arrow heads) in the neoplastic foci of the lumbar spinal cord. The round tumor cells with abundant clear cytoplasm are negative for GFAP. GFAP-immunostain. × 300. following antibodies were used as primary antibodies; rabbit Dako), myelin basic protein (MBP, prediluted, Zymed antisera against human glial fibrillary acidic protein (GFAP, Laboratories, South San Francisco, CA, U.S.A.) and - Prediluted, Dako), bovine galactocerebroside (GC, 1:200, specific enolase (NSE, 1:200, Dako), and mouse monoclonal Chemicon, Temecula, CA, U.S.A.), bovine S-100 (1:400, antibodies against human neurofilament (NF, 1:20, Dako), BOVINE SPINAL OLIGODENDROGLIOMA 1325 vimentin (1:40, Dako), synaptophysin (1:20, Dako), and of the tumor for GFAP, NSE, NF and synaptophysin may proliferating cell nuclear antigen (PCNA, prediluted, Dako). support our diagnosis. A few GFAP-positive astrocytes To visualize microglia, sections were stained with were present in the neoplastic foci, and diffuse proliferation biotinylated lectin Ricinus communis agglutinin-1 (RCA-1, of GFAP-positive astrocytes was recognized at the tumor 1:400, EY Laboratories, San Mateo, CA, U.S.A.). The lectin periphery. These GFAP-positive astrocytes were considered staining was done using the avidin-biotin peroxidase to be reactive astrocytes. The presence of a sub-population complex method (Vector Laboratories, Burlingame, CA, of neoplastic cells with astrocytic differentiation known as U.S.A.). Immunohistochemically, neoplastic round cells minigemistocytes or gliofibrillary has been with abundant clear cytoplasm and small lymphocyte-like demonstrated in many human oligodendrocytic tumors [4, cells were completely negative for GFAP. Only a few of 11]. However, we were unable to confirm the neoplastic GFAP-positive astrocytes were recognized within the nature of the GFAP-positive cells in the present case. Since neoplastic foci (Fig. 6), and many GFAP-positive astrocytes histopathological differentiation between pure were distributed at the periphery. Immunostaining for MBP oligodendroglioma and mixed (oligo-astroglioma) and GC labeled myelin sheaths and intact oligodendrocytes may be quite difficult, mixed glioma could still be intensely, and intact and reactive astrocytes also showed considered in the differential diagnosis of this tumor. moderate immunoreactivity for GC. Neoplastic round cells Anaplastic (malignant) oligodendrogliomas are characterized were moderately positive for GC and negative for MBP. by anaplastic morphology and prominent mitotic activity of The neoplastic cells were negative for other antibodies, tumor cells, vascular endothelial proliferation, and abundant including NF, synaptophysin, vimentin, S-100, NSE, and necrosis sometimes with calcium deposits [9, 12, 13]. In lectin RCA-1. The number of tumor cells with PCNA- spite of the wide arachnoidal dissemination, the tumor cells positive nuclei was very small, approximately 3 to 5 cells showed the typical morphology of oligodendroglioma with under high-power magnification (10 × 40). A similar low growth activity, suggested by the extreme rarity of number of PCNA-positive astrocytes were detected at the mitotic figures and PNCA immunoreactivity. Moreover, periphery of the neoplastic foci. The results of PCNA endothelial proliferation, necrosis, and calcification were not staining indicate a poor growth activity of the neoplastic prominent in the present tumor. Thus, these morphological cells. features are thought to be quite different from those of Typically oligodendroglioma forms soft gelatinous mass, anaplastic oligodendroglioma. occasionally containing cystic spaces and leptomeningial or Although there are few reports of spinal ventricular extension is quite common [2, 8, 14]. oligodendrogliomas in animals [5, 15], the spinal white Histopathologically, this tumor is characterized by a matter may be affected. Even in humans spinal honeycomb appearance formed by accumulation of tumor oligodendrogliomas are described as rare, and most cases cells with artifactualy swollen clear cytoplasm and central have arisen in children [2, 8, 14]. is nuclei [9, 12, 13]. Several immunohistochemical markers considered to be effective for this tumor, but many cases such as MBP, GC, and Leu 7, have been applied for have a poor clinical prognosis. Nam et al. [8] mentioned diagnosis of human oligodendrogliomas [10, 12], although that the poor prognosis of spinal oligodendroglioma might the findings have varied from case to case. These previous be due to its biological nature frequently showing wide studies indicated there were no specific markers for the extension or dissemination. In their review, Fortuna et al. diagnosis of oligodendrogliomas. Moreover, the co- [2] cited several articles in which intracranial or spinal presence of GFAP-positive cells in oligodendrogliomas [4, meningeal extension of oligodendroglioma known as 6] becomes problematic when trying to differentiate pure “oligodendrogliomatosis”, was reported as frequent, seven oligodendroglioma from mixed glioma (oligo-astroglioma). of 10 necropsied cases showing pathological evidence for The present spinal tumor had the histological this event. In oligodendrogliomas of animals, similar characteristics of typical oligodendroglioma reported in biological features have been emphasized [13], although humans and several animal species [9, 12, 13]. The tumor such findings might be based mainly on canine cerebral showed a honeycomb appearance formed by accumulation oligodendrogliomas, including anaplastic of round cells with vacuolated cytoplasm, and these oligodendrogliomas. In the present case, diffuse arachnoidal neoplastic cells were negative for GFAP. Cystic spaces dissemination of the tumor cells was found in the containing small round neoplastic cells, were prominent in subarachnoidal space from the 2nd lumbar to 3rd sacral this tumor. In both human and animal oligodendrogliomas, levels of spinal cord, in spite of the well differentiated similar cystic changes have been commonly described, and morphological features and low mitotic activity. Since are considered to occur by mucinous degeneration of the diffuse invasion of neoplastic cells in the subarachnoidal tumor [9, 12, 13, 15]. Based on these morphological space was recognized at the primary site, tumor seeding features, the present case was diagnosed as might have occurred easily and widely to the lower part of oligodendroglioma. Recently, it has been reported that some the spinal cords through the cerebrospinal fluid. 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